Objective ·To evaluate the diagnostic performance and application value of a domestically developed microfluidic technology-based nucleic acid detection system for gastrointestinal pathogens. Methods ·A total of 534 fecal samples were collected from Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, between April 5th, 2024 and October 6th, 2025. Based on the purpose of testing, the samples were divided into three independent cohorts. Cohort A included 15 samples confirmed positive for Clostridium difficile by the Xpert Clostridiumdifficile assay (Xpert C. difficile), which were used to evaluate the performance of the gastrointestinal pathogen detection panel (high definition gastrointestinal panel, HD GI panel) for detecting Clostridium difficile. Cohort B included 167 samples from patients requiring comprehensive gastrointestinal pathogen screening and was used to compare the multi-pathogen detection performance of the HD GI panel with the FilmArray gastrointestinal panel (FilmArray GI panel). Cohort C consisted of 352 fecal samples from children with clinically suspected gastrointestinal infections who were admitted to the pediatric intensive care unit (PICU), and was used to analyze the epidemiological characteristics of gastrointestinal pathogens in critically ill children and the clinical application value of the HD GI panel. Sociodemographic and baseline clinical data of children in Cohort C were collected. The Kappa test assessed the agreement between the two detection methods. The McNemar test was used to compare the differences in positive detection rates between the two methods. Pearson correlation analysis was used to evaluate the correlation CT values obtained by the two methods. Using the results of the FilmArray GI panel as the reference standard, the sensitivity, specificity, positive predictive value, and negative predictive value of the HD GI panel, along with their 95% confidence intervals, were calculated. Multivariate Logistic regression analysis was used to identify factors affecting the detection accuracy. Results ·The detection results for Clostridium difficile obtained with the HD GI panel and the Xpert C. difficile assay were consistent, and the CT values showed a moderate positive correlation (r=0.594, P=0.019). The overall compliance between the HD GI panel and the FilmArray GI panel was 92.22%, and the Kappa test indicated good consistency between the two methods (κ=0.823, P<0.001). Compared with the FilmArray GI panel, the HD GI panel achieved a sensitivity of 88.89%, a specificity of 93.81%, a positive predictive value of 87.27%, and a negative predictive value of 94.64%. Among critically ill children, the overall positivity rate detected by the HD GI panel was 39.77%. Norovirus GⅡ, rotavirus A, and adenovirus were the predominant viral pathogens, whereas enteroaggregative Escherichia coli (EAEC), Clostridium difficile, and Salmonella were the main bacterial pathogens. Co-infections accounted for 30.00%. Compared with clinical diagnosis, the HD GI panel demonstrated a sensitivity and a negative predictive value of 100.00%, with a diagnostic accuracy of 93.60%. The Kappa test showed a high level of agreement between the two methods (κ=0.860, P<0.001). Multivariate Logistic regression analysis further revealed that the presence of underlying diseases was an independent factor associated with detection accuracy (OR=0.051, 95%CI 0.014‒0.141, P<0.001). Conclusion ·The HD GI panel demonstrates diagnostic performance comparable to that of mainstream platforms for gastrointestinal pathogen detection, offering advantages such as simple operation, rapid turnaround, and a pathogen spectrum consistent with the characteristics of pediatric gastrointestinal infections in China. It is well suited for use in primary hospitals and high-throughput testing settings.